Differential expression of the two types of histone H2A genes in wheat.

Five histone H2A cDNA clones have been isolated from a wheat cDNA library. They were divided into two groups, termed type 1 and type 2, based on their deduced amino acid sequences and their gene expression patterns. Three type 1 clones had ORFs encoding proteins similar to angiosperm histone H2As known so far, whereas two type 2 clones encoded an identical protein, which was more similar to Norway spruce (gymnosperm) H2A than to the angiosperm H2As. The C-terminus of the type 2 H2A was shorter than that of the type 1 H2As and lacked the characteristic SPKK motif that is conserved in angiosperm H2As. Northern analysis revealed that the mRNA levels of the type 1 H2A genes were high in proliferating cells during germination and in various tissues of young seedlings, while the mRNA levels of the type 2 genes were high in non-proliferating cells in which the type 1 gene was poorly expressed. This result suggests that the expression of these two groups of H2A genes is differently regulated during development in wheat.

The putative zinc-finger protein WZF1 interacts with a cis-acting element of wheat histone genes.

A nonamer motif (CATCCAACG) that is one of the cis-acting elements identified in the proximal promoter region of some wheat histone genes is included in the region that interacts with the wheat DNA-binding protein, HBP (histone gene-binding protein)-2. To obtain structural and functional information about this DNA-binding protein, we attempted to isolate a cDNA clone encoding HBP-2 on the basis of its ability to bind to a nonamer-containing 38-bp DNA fragment. Southwestern screening of a wheat cDNA library with concatenated 38-residue oligonucleotides as the probe produced one candidate clone. Nucleotide sequence analyses of this cDNA clone and the corresponding genomic clone showed that the protein deduced from the nucleotide sequence consisted of 261 amino acids and contained a set of zinc-finger motifs similar to those found in many eukaryotic transcription factors. The protein, named WZF1 (wheat zinc-finger protein 1), which was expressed from the cDNA in Escherichia coli cells, bound specifically and metal-ion-dependently to the nonamer-containing oligonucleotide. The WZF1 mRNA was highly expressed in the root apexes of wheat seedlings, but less so in the proximal portion of young leaves; whereas, histone H3 mRNA was highly expressed in both tissues. The expression patterns of the WZF1 and histone H3 genes in the early stages of germination differed, expression of the WZF1 gene being almost constant but not that of the H3 gene. The relationship of WZF1 and HBP-2 and the possible role of WZF1 in the histone gene expression were discussed.

Coordinate gene expression of five subclass histones and the putative transcription factors, HBP-1a and HBP-1b, of histone genes in wheat.

The expression of genes encoding five histones (H1, H2A, H2B, H3 and H4) and the putative transcription factors HBP-1a (17) and HBP-1b (c38) was examined during early germination and in various tissues of young wheat seedlings. The steady-state levels of core histone (H2A, H2B, H3 and H4) mRNAs were coordinately cell cycle-dependent and paralleled the rate of DNA synthesis during early germination, whereas the expression pattern of the linker histone (H1) genes differed. The five subclass histone genes were actively expressed in the meristematic tissues of young seedlings. Moreover, H1 genes were expressed in leaves that consist mostly of non-proliferating cells, in which core histone genes showed little expression. Quantitative alterations to the mRNAs of the putative transcription factors HBP-1a (17) and HBP-1b (c38) of wheat histone genes were similar to those of the core histone mRNAs, suggesting that both factors function in the cell cycle-dependent expression of wheat core histone genes.

Structural characteristics of two wheat histone H2A genes encoding distinct types of variants and functional differences in their promoter activity.

To investigate the regulation of plant histone H2A gene expression, we isolated two H2A genes (TH254 and TH274) from wheat, which encode two variants of H2A. Both genes had an intron in the coding region. In the promoters, some characteristic sequences, such as Oct and Nona motifs, which are conserved among plant histone genes, were located in a short region (about 120 bp) upstream from the putative TATA box. Transient expression analyses of promoter activity with H2A-GUS fusion genes using tobacco protoplasts revealed novel types of positive cis-acting sequences in the TH254 promoter: a direct repeat of a 13 bp sequence (AGTTACATTATTG) and a stretch composed of an AT-rich sequence (ATATAGAAAATTAAAA) and a G-box (CACGTG). Quantitative S1 assay of the mRNA amounts from the TH254/GUS and TH274/GUS chimeric genes in stably transformed and cell cycle-synchronized tobacco cell lines showed that the promoters of both genes contained at least one cis-acting element responsible for S phase-specific expression. Histochemical analysis of transgenic tobacco plants carrying the chimeric genes showed that the promoters of the two H2A genes were active in developing seedlings and flower organs but were regulated in a different manner.

Molecular cloning and characterization of cDNAs for anionic and neutral peroxidases from suspension-cultured-cells of sweet potato and their differential expression in response to stress.

Two peroxidase (POD) cDNAs, swpal and swpn1, were isolated and characterized from suspension-cultured cells of sweet potato in order to understand the physiological function of POD isozymes. Sequence analysis showed that swpa1 encoded an anionic POD and swpn1 encoded a neutral POD. The swpa1 and swpn1 genes were both highly expressed in suspension-cultured cells in accordance with the high POD activity of these cells. Although both gene transcripts were detected in the stems of intact plants, their transcription levels were much lower than in suspension-cultured cells. During cell growth the pattern of mRNA accumulation of swpa1 differed from that of swpn1, suggesting that expression of these genes is differentially regulated by cell growth stage. In addition, the swpa1 and swpn1 genes responded differently to oxidative stress induced by chilling. The expression of swpa1 was weakly induced by 15 degrees C acclimation and strongly induced by 4 degrees C chilling, whereas the mRNA level of swpn1 was increased by 15 degrees C acclimation and reduced by 4 degrees chilling. This indicates that the two isozymes encoded by swpa1 and swpn1 might contribute to protection against cold-induced oxidative stress through different signaling pathways. In leaves, both genes were induced by wounding with broadly similar expression. patterns. Genomic analysis suggests that the two isozymes are encoded by different loci in the sweet potato genome.

Molecular characterization of cDNAs for two anionic peroxidases from suspension cultures of sweet potato.

Two cDNAs for anionic peroxidase (PODs), swpa2 and swpa3, were isolated from suspension cultures of sweet potato (Ipomoea batatas), and their expression was investigated with a view to understanding the physiological function of PODs in relation to environmental stresses. Swpa2 (whose putative mature protein product would have a pI value of 4.1) and swpa3 (4.3) encode polypeptides of 358 and 349 amino acids, respectively. The genes from which they were derived are predominantly expressed in cultured cells of sweet potato; transcripts of swpa2 were not detected in any tissues of the intact plant, and transcripts of swpa3 were detected at a low level only in the stem tissue. During cell culture, the expression patterns of the two genes differed; the level of swpa2 RNA progressively increased during cell growth, whereas that of swpa3 reached a maximum at the stationary phase and decreased on further culture. The two genes responded differently to stresses such as wounding or chilling of leaves. Swpa2 was strongly induced 48 h after wounding, but swpa3 was not affected by this treatment. The two genes were also highly expressed upon chilling (4 degrees C), but expression was reduced by prior acclimation at 15 degrees C. In addition, both genes were strongly induced immediately after treatment with ozone, and expression had decreased to the basal level 12 h after treatment. The response of these two genes to stresses such as aging, wounding, and chilling are different from those of the POD genes (swpa1 encoding an anionic product and swpn1 a neutral peroxidase) that we described previously. The responses of the two genes were also different from each other. These results suggest that the two new POD genes are involved in overcoming oxidative environmental stress, and each POD gene may be regulated by cell growth and environmental stress in different ways.

A nitrilase-like protein interacts with GCC box DNA-binding proteins involved in ethylene and defense responses.

Ethylene-responsive element-binding proteins (EREBPs) of tobacco (Nicotiana tabacum L.) bind to the GCC box of many pathogenesis-related (PR) gene promoters, including osmotin (PR-5). The two GCC boxes on the osmotin promoter are known to be required, but not sufficient, for maximal ethylene responsiveness. EREBPs participate in the signal transduction pathway leading from exogenous ethylene application and pathogen infection to PR gene induction. In this study EREBP3 was used as bait in a yeast two-hybrid interaction trap with a tobacco cDNA library as prey to isolate signal transduction pathway intermediates that interact with EREBPs. One of the strongest interactors was found to encode a nitrilase-like protein (NLP). Nitrilase is an enzyme involved in auxin biosynthesis. NLP interacted with other EREBP family members, namely tobacco EREBP2 and tomato (Lycopersicon esculentum L.) Pti4/5/6. The EREBP2-EREBP3 interaction with NLP required part of the DNA-binding domain. The specificity of interaction was further confirmed by protein-binding studies in solution. We propose that the EREBP-NLP interaction serves to regulate PR gene expression by sequestration of EREBPs in the cytoplasm.

Stress signaling through Ca2+/calmodulin-dependent protein phosphatase calcineurin mediates salt adaptation in plants.

Calcineurin (CaN) is a Ca2+- and calmodulin-dependent protein phosphatase (PP2B) that, in yeast, is an integral intermediate of a salt-stress signal transduction pathway that effects NaCl tolerance through the regulation of Na+ influx and efflux. A truncated form of the catalytic subunit and the regulatory subunit of yeast CaN were coexpressed in transgenic tobacco plants to reconstitute a constitutively activated phosphatase in vivo. Several different transgenic lines that expressed activated CaN also exhibited substantial NaCl tolerance, and this trait was linked to the genetic inheritance of the CaN transgenes. Enhanced capacity of plants expressing CaN to survive NaCl shock was similar when evaluation was conducted on seedlings in tissue culture raft vessels or plants in hydroponic culture that were transpiring actively. Root growth was less perturbed than shoot growth by NaCl in plants expressing CaN. Also, NaCl stress survival of control shoots was enhanced substantially when grafted onto roots of plants expressing CaN, further implicating a significant function of the phosphatase in the preservation of root integrity during salt shock. Together, these results indicate that in plants, like in yeast, a Ca2+- and calmodulin-dependent CaN signal pathway regulates determinants of salt tolerance required for stress adaptation. Furthermore, modulation of this pathway by expression of an activated regulatory intermediate substantially enhanced salt tolerance.